An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500-fold from Micrococcus luteus. In this purification, separation from the pyrimidine-dimer-specific ultraviolet-endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg", but 5-30 mM Mg2' produces a five-fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, b-propiolactone, 7-bromomethyl-benz [a]anthracene, N-acetoxy-2-acetylaminofluorene, and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action.The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel electrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms.A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e. apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites'present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme-catalyzed singlestrand breaks found in ultraviolet-irradiated DNA was five times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized in addition to apurinic sites.It is well established that the exposure of DNA to ultraviolet light, ionizing radiation, mutagens and carcinogens may lead to DNA damage. Ultraviolet light induces, predominantly, pyrimidine dimers [l] ; the most readily detectable DNA alterations arising from ionizing radiation are single-strand breaks and Abbreviations. MeSOZOMe, methyl methanesulfonate; ethyl methanesulfonate; MeNOUr, EtNOUr, BuNOUr, PeNOUr, MezS04, dimethyl sulfate; Et2SO4, dielhyl sulfate; nitrogen mustard, 2,2'-dichloro-Nmethyldiethylamine hydrochloride; (Ac)lONFln, N-acetoxy-2-acet y laminofluorene. double-strand breaks [2], whereas mutagens and carcinogens modify DNA by transferring electrophilic chemical residues [3 -51 which might cause steric alterations [6] and/or loss of bases [7,8].Living cells, mammalian or bacterial, respond...

A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a functiW of enzyme concentration follows a sigmoida}+curve. The enzyme requires Mg for activity. In the presence of 4.5 mM Mg addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates age also obsuved in the absence of KCl, when the reaction is carried out at 0 C or at Mg concentrations exceeding 10 mM. INTRODUCTIONDNA-relaxing enzymes catalyze the conversion of superhelical DNA to a non-superhelical covalently closed form (1-4) presumably by introducing a transient swivel into the helix (5). The relaxation process probably does not follow a single-hit mechanism as intermediates are generated having smaller numbers of superhelical turns than the original DNA substrate (3,5,6). It seems accepted that the essential steps of the DNA relaxation involve breakage of one strand, winding of that strand relative to the other and sealing of the break (5,7). The in vivo functions of DNA-relaxing enzymes are still uncertain. The enzymes may be involved in any process requiring winding or unwinding of the double helix such as replication (8) or transcription (9). DNA-relaxing activities have been purified from Escherichia coli (1,10), vaccinia virus (11), Drosophila melanogaster (3), yeast (Thielmann and Hess, in preparation), calf thymus (12), rat liver (13), mouse embryo cells (4), HeLa (4) and KB cells (6). The enzymes of eukaryotic origin are distinguished

Superhelical DNA of the Pseudomonas phage PM2 was irradiated with UV-light or reacted with covalently binding carcinogens, such as 7-bromomethyl-benz[a]anthracene, (Ac)2ONFln, K-region epoxides, and alkylating agents. Migration velocity of the DNA products was determined using agarose gel electrophoresis. In gels of more than 1.3%-1.9% agarose, modified PM2 DNA exhibited a dose-(concentration-)dependent decrease of migration velocity. This phenomenon is probably due to a decrease in superhelix density which caused the compact DNA coil to assume eventually an open-circular conformation. Comparison of the extent of DNA modification with the decrease of migration velocity revealed that the superhelical structure sensitively reflected the chemical DNA alterations. DNA species exhibiting, in 1.6% agarose gels, a migration velocity of up to 30% of that of control DNA showed an increase of velocity in 0.4% agarose. Therefore, in 1.3%-1.9% agarose gels, the decrease os superhelix density is accompanied by an increase of the frictional coefficient, whereas in 0.4%-0.9% agarose gels the same decrease of superhelix density apparently led to a higher degree of flexibility of the macromolecule and/or exposure of additional electric charges.